The encapsulated fungus Cryptococcus neoformans is responsible for life- threatening disease, particularly in the context of compromised immunity, and current therapy is not adequate. Extracellular glycans define the interface between this opportunistic pathogen and its host;our long-term goal is to determine the biosynthetic pathways of these molecules and discover ways to interfere with them. The main virulence factor of C. neoformans is an extensive polysaccharide capsule composed of two polymers: glucuronoxylomannan (GXM) and glucuronoxylomanno-galactan (GXMGal;previously termed GalXM). We know the structures of these polysaccharides, but major questions remain concerning their synthesis and assembly into the capsule. In the preceding period of this project we purified, cloned, expressed, and mutated a novel xylosyltransferase (Cxt1p), and showed that it acts in synthesis of GXM, GXMGal, and glycolipids;we also discovered a related enzyme (Cxt2p). We further established how capsule is transported to the cell surface and demonstrated that cell wall alpha glucan is required for capsule display. Aim I of the current application is to localize Cxt1p and Cxt2p within the cryptococcal cell and perform targeted experiments on glycan structure and virulence to address their role in cryptococcal biology. Aim II proposes a biochemical approach to identify the machinery of capsule association with the cell surface, followed by phenotypic analysis of strains deficient in candidate proteins. These revised aims can be reasonably performed in two years. We plan creative new strategies to investigate novel features of C. neoformans glycobiology, with emphasis on the capsule. We will capitalize on our prior discoveries to gain greater understanding of cryptococcal biology and pathogenesis, identify targets for anti-fungal drug discovery, and elucidate glycan synthesis in eukaryotes.